The invention concerns oligosaccharide mixtures having antithrombotic activity and is particularly concerned with oligosaccharides derived from dermatan sulphate.
Dermatan sulphate is a linear polysaccharide which has antithrombotic activity and is composed of alternating glucuronic or iduronic acid residues and N-acetylgalac-tosamine residues, these residues being sulphated to a varying extent. The mechanism of its activity in the inhibition of thrombin depends on the dermatan sulphate binding to both heparin cofactor II (HCII, a serine protease inhibitor) and to thrombin (factor IIa) itself.
Numerous studies have been made in the past of dermatan sulphate and its degradation products and these studies have mostly been directed to the identification and isolation of specific fragments having activity of interest. Thus for example J. Biological Chemistry, vol. 261, July 1986, 8854-58 describes fragments containing 12-14 sugar residues which bind to HCII and the same article also describes di-, tetra-, and hexasaccharide fragments without this binding ability. J. Biological Chemistry, vol. 265, October 1990, 18263-71 on the other hand describes hexasaccharides with a high binding affinity to HCII. Oligosaccharides containing 5 or 6 sugar residues and extended forms which bind to HCII are described in WO 91/15217. Thrombosis Research, 66, 1992, 527-33 suggests that the best balance of biological activity, clearance, half-life of disappearance, bioavailability, and other factors is achieved with oligosaccharides having molecular weights ranging from 4-9 kDa. WO 93/05074 describes oligosaccharides with antithrombotic activity which contain 11-13 sugar residues, and WO 93/05075 describes oligosaccharides having 7-12 sugar residues. Blood, vol. 81, April 1993, 1771-77 concludes that the fragment of minimum size for full catalytic activity is a hexadecasaccharide.
The methods used in the earlier investigations to produce the fragments studied generally involve degradation of the dermatan sulphate starting material and isolation of the fragments by affinity chromatography.
A native low molecular weight dermatan sulphate (Desmin) currently under investigation is characterised by a sulphate to carboxylate ratio of 1.06 (Thrombosis Research, vol. 83, no. 1, 103-109).
It will be appreciated from the above that numerous oligosaccharide fragments can be obtained from dermatan sulphate and the relationship between the nature of these oligosaccharides and their activity is complex and has not yet been completely elucidated.
We have now found that a satisfactory level of antithrombotic activity can be found in fractions which have been separated from material with a lower charge density and contain mixtures of oligosaccharides based on specific disaccharide residues.
The oligosaccharides of the invention are different from prior art since they are more negatively charged. They can be prepared by methods which are commercially more competitive compared to those previously used and described in the prior art proposals in the isolation of specific fragments, since they are less expensive to manufacture. These dermatan sulphate fractions show surprisingly higher affinity towards HQII than native dermatan sulphate and can therefore be used to increase the antithrombotic potency of dermatan sulphate based drugs.
The invention thus provides a mixture of linear oligosaccharides containing different numbers of repeating sulphated disaccharide units derived from L-iduronic acid (IdoA) and N-acetyl-D-galactosamine (Gal-NAc) and having the following characteristics:
(a) a molecular weight in the range of 1600-20000 for 90% or more of the mixture,
(b) a sulphur content of 6.0-8.0% by weight,
(c) a sulphate/carboxylate ratio of 1.2-1.6,
(d) a disulphated disaccharide content of 20-60% by weight of the mono-sulphated disaccharide content, and
(e) an antithrombin activity of 20-60 IU/mg.
The mixture is prepared from dermatan sulphate and may thus contain a small proportion of residues derived from D-glucuronic acid, although it is preferably free or essentially free of such residues. Residues of hexosamines other than Gal-NAc may also be present in a small amount, e.g. residues of N-acetyl-D-glucosamine (Glu-NAc) although Gal-NAc is preferably the only hexosamine present.
The Gal-NAc residues are sulphated at the 4-position in most cases and the IdoA residues are sulphated at the 2-position in some instances. Some cases of sulphation at the 6-position of Gal-NAc are also seen. The sul-phate:carboxyl and disulphated:monosulphated disaccharide ratios and the sulphur content can thus vary, mostly depending on the extent of sulphation of the IdoA residues.
Formula 1 shows the sequence of repeating sulphated L-iduronic acid and N-acetyl-D-galactosamine units generally present in the oligosaccharides of the invention. 
where R is H or SO31 3, and
n is in the range 3-37 for 90% or more of the material and 10-15 for the peak point of the molecular distribution.
The residues are linked from the IdoA 1-position tothe Gal-NAc 3-position and from the Gal-NAc 1-position to the IdoA 4-position. When prepared by the method described below, a proportion of the terminal Gal-NAc residues have a 2,3,4-trihydroxybutyric acid residue (HOCH2xe2x80x94CH(Oxe2x80x94)xe2x80x94CH(OH)COOxe2x88x92) at the 1-position. The counterion is a mono- or divalent cation, mostly sodium but possibly also other ions such as calcium.
The properties characteristic of the oligosaccharides may be measured by the following methods:
(1) molecular weight by gel permeation chromatography (GPC)xe2x80x94HPLC according to the method of Dedem and Nielsen, Pharmeuropa Vol. 3, No. 3, 202-218 (1991),
(2) sulphur content according to Ph. Eur. V.3.5.3,
(3) sulphate/carboxyl ratio according to Ph. Eur. p. 828,
(4) disulphated/monosulphated disaccharide content by the method of Linhardt et al., Anal. Biochem. 181, 288-296 (1989).
(5) HCII-mediated antithrombin activity by a chromogenic assay (Diagnostica Stago, France) in a plasma free system with the 4. International Heparin Standard (code no. 82/502) as standard.
The sulphur content of the mixtures is generally within the range 6.0-8.0% by weight and is preferably 6.5-8.0%. The sulphate/carboxyl ratio can generally range from 1.2-1.6 and is preferably from 1.3-1.6. The content of disulphated disaccharide is usually from 20-60% by weight of the monosulphated disaccharide content and is preferably 30-60% of the latter. The HCII mediated antithrombin activity of the oligosaccharides of the invention is generally in the range 20-60 IU/mg and preferably 30-60 IU/mg.
The oligosaccharide mixture of the invention may be prepared by depolymerisation of dermatan sulphate by periodate oxidation, followed by borohydride reduction and acid hydrolysis and then ion exchange fractionation.
The dermatan sulphate used as the starting material may be derived from animal intestines such as for example porcine mucosa or bovine trachea and have the following characteristics:
Specific optical rotation (4% in water): xe2x88x9250xc2x0 to xe2x88x9270xc2x0
Sulphur: 5.3-6.3% (w/w)
Sulphate/carboxylate ratio: 1.0-1.3
HCII mediated antithrombin activity: 2-10 IU/mg
The main chemical reactions involved in the depolymerisation process are shown in the reaction scheme below and principally follow the route described in Carbohydrate Research 36, 339-348 (1974).
Depolymerisation of dermatan sulphate: 
In the first step, treatment of a dermatan sulphate (1) with periodate results in selective oxidation of non-sulphated IdoA residues to produce a ring-opened dialdehyde (2). Reduction of the latter gives the diol (3) which is then cleaved by acid hydrolysis to give two Gal-NAc-terminated fragments, one of which carries a 2,3,4-trihydroxybutyric acid end group. This cleaving occurs at numerous positions along the dermatan sulphate chain, and gives a mixture of fragments of different chain lengths. A selected fraction of these fragments is then obtained by ion exchange fractionation.
The initial oxidation step may be carried out with periodic acid or a salt thereof, for example the sodium salt, in an aqueous medium at a temperature of 0-30xc2x0 C. and a pH of 5-8. The reduction step may be carried out with a borohydride such as potassium or, preferably, sodium borohydride, in an aqueous medium at a temperature of 0-30xc2x0 C. and a pH of 6-9. The acid hydrolysis step is preferably carried out with a mineral acid such as hydrochloric or sulphuric acid at any suitable temperature, e.g. 20-60xc2x0 C.
The ion exchange fractionation of the mixture generally separates relatively highly sulphated fragments from fragments having a lower degree of sulphation, where the highly sulphated fractions are characterised by
(a) a molecular weight in the range of 1600-20000 for 90% or more of the mixture,
(b) a sulphur content of 6.0-8.0% by weight,
(c) a sulphate/carboxylate ratio of 1.2-1.6,
(d) a disulphated disaccharide content of 20-60% by weight of the mono-sulphated disaccharide content, and
(e) an antithrombin activity of 20-60 IU/mg.
The fractionation may be carried out by stepwise eluation using an ion exchange resin such as Amberlite IRA 404, Amberlite IRA 900, Amberlite IRA 904, Amberlite IRA 958, Amberlite IRA 67, Amberlite IRA 68, Lewatit S5428A, Lewatit S6328A, Lewatit S6328, Lewatit MP 500WS, Dowex 11, Dowex Monosphere 550A, Dowex MSA1, or any other anion exchanger.
The ion exchange step may be carried out by binding a solution of the starting material to the resin and subsequently eluting fractions with different activity from the resin depending on the ionic strength of the eluation solution. Low sulphated low molecular weight products are usually eluted first and discarded, and the required highly sulphated low molecular weight material is then obtained by further elution.
The oligosaccharide mixtures of the invention can be formulated as pharmaceutical compositions for use in human or veterinary medicine as antithrombotic agents in any convenient way. Such compositions will usually include one or more acceptable carriers and may for example be in a form suitable for parenteral, oral, or topical use.
The mixtures may be formulated for use in veterinary or human medicine by injection and may be presented in unit dose form, e.g. in ampoules. The compositions for injection may be in the form of suspensions, solutions, or emulsions,. in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising, solubilising, and/or dispersing agents.
The compositions may also be in a form suitable for oral administration, for example in the form of solutions, syrups, or suspensions, or a dry powder for constitution with water or other suitable vehicle before use. Solid compositions such as tablets, capsules, lozenges, pills, boluses, powder, pastes, granules, bullets, or premix preparations may also be used.
The composition may also be in a form suitable for topical administration, for use in veterinary and human medicine, e.g. ointments, creams, lotions, shampoos, powders, sprays, dips, or aerosols.
The mixtures of the invention may be administered in combination with other pharmaceutically active ingredients.
The total daily dosage of mixtures of the invention employed in both veterinary and human medicine will suitably be in the range of 1-100 mg/kg bodyweight, preferably from 3-30 mg/kg and these may be given in divided doses, e.g. 1-4 times per day.
Formulations of the invention can be used for treatment of different thrombotic conditions such as deep venous thrombosis (DVT) and stroke as well as for prophylaxis in general surgery, for example hip replacements and knee operations to prevent thrombotic complications.